The present invention relates to a newly identified receptor belonging to the superfamily of G-protein-coupled receptors. The invention also relates to polynucleotides encoding the receptor. The invention further relates to methods using the receptor polypeptides and polynucleotides as a target for diagnosis and treatment in receptor-mediated disorders, specifically, cardiovascular diseases, including congestive heart failure. The invention further relates to drug-screening methods using the receptor polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the receptor polypeptides and polynucleotides. The invention further relates to procedures for producing the receptor polypeptides and polynucleotides.
G-protein coupled receptors (GPCRS) constitute a major class of proteins responsible for transducing a signal within a cell. GPCRs have three structural domains: an amino terminal extracellular domain, a transmembrane domain containing seven transmembrane segments, three extracellular loops, and three intracellular loops, and a carboxy terminal intracellular domain. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs.
GPCR genes and gene-products are potential causative agents of disease (Spiegel et al., J. Clin. Invest. 92:1119-1125 (1993); McKusick et al., J. Med. Genet. 30:1-26 (1993)). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans et al., Annu. Rev. Genet. 26:403-424(1992)), and nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2:1201-1204 (1993)). These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
The GPCR protein superfamily can be divided into five families: Family I, receptors typified by rhodopsin and the xcex22-adrenergic receptor and currently represented by over 200 unique members (Dohlman et al., Annu. Rev. Biochem. 60:653-688 (1991)); Family II, the parathyroid hormone/calcitonin/secretin receptor family (Juppner et al., Science 254:1024-1026 (1991); Lin et al., Science 254:1022-1024 (1991)); Family III, the metabotropic glutamate receptor family (Nakanishi, Science 258 597:603 (1992)); Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al., Science 241:1467-1472 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, Annu. Rev. Biochem. 61:1097-1129 1992)).
There are also a small number of other proteins which present seven putative hydrophobic segments and appear to be unrelated to GPCRs; they have not been shown to couple to G-proteins. Drosophila expresses a photoreceptor-specific protein, bride of sevenless (boss), a seven-transmembrane-segment protein which has been extensively studied and does not show evidence of being a GPCR (Hart et al., Proc. Natl. Acad. Sci. USA 90:5047-5051 (1993)). The gene frizzled (fz) in Drosophila is also thought to be a protein with seven transmembrane segments. Like boss, fz has not been shown to couple to G-proteins (Vinson et al., Nature 338:263-264 (1989)).
G proteins represent a family of heterotrimeric proteins composed of xcex1, xcex2 and xcex3 subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane segments. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the xcex1-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the xcex2xcex3-subunits. The GTP-bound form of the xcex1-subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates. Greater than 20 different types of a-subunits are known in humans. These subunits associate with a smaller pool of xcex2 and xcex3 subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish et al., Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins and G protein-linked effector and second messenger systems have been reviewed in The G-Protein Linked Receptor Fact Book, Watson et al., eds., Academic Press (1994).
GPCRs are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs. The present invention advances the state of the art by providing a previously unidentified human GPCR.
It is an object of the invention to identify novel GPCRs.
It is a further object of the invention to provide novel GPCR polypeptides that are useful as reagents or targets in receptor assays applicable to treatment and diagnosis of GPCR-mediated disorders.
It is a further object of the invention to provide polynucleotides corresponding to the novel GPCR receptor polypeptides that are useful as targets and reagents in receptor assays applicable to treatment and diagnosis of GPCR-mediated disorders and useful for producing novel receptor polypeptides by recombinant methods.
A specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the novel receptor.
A further specific object of the invention is to provide compounds that modulate expression of the receptor for treatment and diagnosis of GPCR-related disorders.
The invention is thus based on the identification of a novel GPCR, designated the 14273 receptor.
The invention provides isolated 14273 receptor polypeptides including a polypeptide having the amino acid sequence shown in SEQ ID NO 1 (human) and SEQ ID NO 4 (murine), or the amino acid sequence encoded by the cDNA deposited as ATCC No. PTA-1143 on Jan. 5, 2000 (xe2x80x9cthe deposited cDNAxe2x80x9d).
The invention also provides isolated 14273 receptor nucleic acid molecules having the sequence shown in SEQ ID NO 2 (human) and SEQ ID NO 5 (murine) or in the deposited cDNA.
The invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO 1 or SEQ ID NO 4 or encoded by the deposited cDNA.
The invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO 2 or SEQ ID NO 5 or in the deposited cDNA.
The invention also provides fragments of the polypeptide shown in SEQ ID NO 1 or SEQ ID NO 4 and nucleotide shown in SEQ ID NO 2 or SEQ ID NO 5, as well as substantantially homologous fragments of the polypeptide or nucleic acid.
The inventors have linked expression of the receptor polynucleotides to cardiovascular disease and specifically to congestive heart failure. The inventors have found that receptor mRNA is induced in hypertrophic human cardiac myocytes and that expression correlates with morphological change. This induction is observed in ischemic and dilated hearts.
Therefore, the invention also provides receptor variants that correlate with the cardiovascular disorders.
Cardiac hypertrophy is the principal response of the heart to overload from any cause including ischemia/reperfusion injury, myocardial infarction, longstanding heart failure, vascular wall remodeling, ventricular remodeling, dilated cardiomyopathy, rapid ventricular pacing, coronary microembolism, pressure-overload, aortic banding, coronary artery ligation, end stage heart failure, tachyarrhythmia, bradyarrhythmia, valvar heart disease, and hypertension. Hypertrophy is a strong, independent predictor of cardiovascular death and is associated with diastolic dysfunction. Since adult cardiac myocytes are terminally differentiated cells, the increase in muscle mass seen in cardiac hypertrophy occurs predominantly by an increase in myocyte size. At the cellular level, the events leading to cardiac hypertrophy have been divided into (1) extracellular hypertrophic stimulus; (2) intracellular signal transduction; and (3) activation of nuclear events that allow for the hypertrophic phenotype.
Further experiments by the inventors have shown that in transgenic mouse models in which the novel receptor is overexpressed, the induction of cardiac hypertrophy is observed. Histological examination of hearts derived from these transgenic mice shows a significant increase in the heart:body weight ratio. In several independent sets of transgenic animals, enlargement of the heart and heart failure is a significant outcome. Observations include enlarged cardiomyocytes, biventricular dilatation, atrial thrombi, and interstitial fibrosis.
Additional experiments have shown that the 14273 receptor was upregulated in a rat pressure overload model. A rat pressure overload model was generated by placing a constrictive band around the aorta to induce a state of cardiac hypertrophy (Kimura et al. Am. J. Physiol. 256:H1006-H1011(1989)). The 14273 receptor was shown to be induced at the mRNA level following banding of the aorta. This increase in expression was correlated with the hypertrophic phenotype. Results were obtained by in situ hybridization.
Additional experiments showed that in transgenic mice overexpressing the 14273 receptor, that receptor mRNA and protein levels were correlated with the severity of the phenotype. The phenotype seen in the 14273 transgenic animals ranged from severe, with death by 3-4 weeks, to mild, showing only subtle pathohistological findings, as evidenced by staining cardiac myocytes from wild-type and transgenic animals with wheat germ agglutinin or Masson""s Trichrome. The wheat germ agglutinin outlines myocytes, which in turn allows the determination of the degree of hypertrophy, while the Trichrome identifies collagen fibers in tissue sections. Collagen fibers are a hallmark of fibrosis. The severity of the phenotype was correlated with increased transgene expression at both the mRNA and protein levels. Severity of the phenotype was proportional to the level of 14273 receptor expression observed. In these experiments hypertrophy was observed in both the nucleus and cytoplasm of the hypertrophic myocytes. In addition, histological results from these animals showed myocyte disorganization, as well as atrial and ventricular loss. This was then associated with subsequent interstitial fibrosis as a result of the replacement of dead myocytes with connective tissue. Fibroelastosis (deficiency in elastic fiber) was also observed. This is evidence of a loss of general elastic tone from cardiac tissue. Finally, atrial thrombosis was observed to result from the above pathological series of events. In addition, along with increased expression of the 14273 receptor, the inventors observed coordinate increased expression of atrial natriuretic factor (ANF). This factor, a natural diuretic, is produced in increased amounts during cardiac hypertrophy and, accordingly, serves as a marker of cardiac hypertrophy.
The invention also provides vectors and host cells for expressing the receptor nucleic acid molecules and polypeptides and particularly recombinant vectors and host cells. The invention particularly provides host cells that provide a model for cardiovascular diseases by containing specific variants that are correlated with the disorders.
The invention also provides methods of making the vectors and host cells and methods for using them to produce the receptor nucleic acid molecules and polypeptides.
The invention also provides antibodies that selectively bind the receptor polypeptides and fragments.
The invention also provides methods of screening for compounds that modulate the activity of the receptor polypeptides. Modulation can be at the level of the polypeptide receptor or at the level of controlling the expression of nucleic acid (RNA or DNA) expressing the receptor polypeptide.
The invention also provides a process for modulating receptor polypeptide activity, especially using the screened compounds, including to treat conditions related to expression of the receptor polypeptides.
Since the receptor polynucleotides have been linked to cardiovascular diseases, the invention provides methods for modulating receptor polypeptide and nucleic acid expression in subjects having, or predisposed to having, cardiovascular diseases, cells from these subjects, and model systems for the disorders.
The invention also provides diagnostic assays for determining the presence of and level of the receptor polypeptides or nucleic acid molecules in a biological sample.
The invention also provides diagnostic assays for determining the presence of a mutation in the receptor polypeptides or nucleic acid molecules.
Since the receptor polynucleotides have been linked to cardiovascular diseases, the invention provides diagnostic assays for determining the presence, level, or mutation of receptor polynucleotides and polypeptides preferably in subjects with, or having a predisposition to, cardiovascular diseases, cells from these subjects, and model systems for the disorders.